This invention relates to droplet flow cytometers such as are used to sort and analyze substances in both clinical and research applications. Specifically, the invention relates to components for such devices which control the differential charging of the droplets for deflection to sort them in the presence of an electrostatic field.
Droplet flow cytometers have been in clinical and research use for many years. Basically, the systems act to position small amounts of a substance within a sheath fluid. Through hydrodynamic focusing and laminar flow, the substance is split into individual cells and the like and is surrounded by the sheath fluid. This sheath fluid may be oscillated and allowed to exit a nozzle in a jet so as to regularly form droplets through the action of surface tension. Since the actual formation of the droplets takes some time after the sheath fluid exits the nozzle, the application of charge to individual droplets must be delayed from the actual time the substance exits the nozzle. This delay must be actually controlled so that when the droplets fall they may be deflected in an electrostatic field. Thus, in order to differentially charge individual droplets, very precise and uniform conditions are desirable within the jet. Unfortunately, variations exist. Thus, accurate processing requires not only properly ascertaining the appropriate time delay in the first place, but also periodically adapting to the changes practically experienced.
In establishing an appropriate droplet formation time delay estimate, two approaches seem to have been taken. The first is to utilize the theoretical predictions. While this approach represents an excellent first approximation, it has the significant shortcoming of not allowing for the variations which inherently exist in an operational system. Some adjustment remains necessary in order to adapt the theoretically predicted time delay to the actual time delay existing in a given system during a given run.
A second approach which appears to have been taken is that mentioned in related U.S. Pat. Nos. 4,317,520, 4,318,480, 4,318,481, 4,318,482, 4,318,483, and 4,325,483. Although not the inventive subject of each of the references, each explains that those skilled in the art had set the time delay by utilizing a test sample which was sorted. The time delay was then manually adjusted until the expected number of target cells was achieved. Obviously, this approach does not take into consideration variations which exist when actually processing. Not only does it require interruption of the processing procedure, but it also ignores the fact that merely changing the substance from a test sample to an actual specimen can introduce changes (such as temperature changes, particulates, bubbles, and the like) which can significantly affect the delay time actually experienced for the formation of the droplets. It also requires manual intervention and thus makes the processing operation less automatic, more personnel intensive, and more susceptible to error.
As a result of the limitations which have appeared to exist in the prior efforts to ascertain a charging time delay, those skilled in the art have apparently been directed away from the aspect of properly setting the time delay and have instead pursued systems which are designed to control the uniformity of the jets so as to cause drop formation to occur at one location without any variation. This type of approach is demonstrated in the five related patents cited earlier as well as two other patents, related U.S. Pat. Nos. 4,487,320 and 4,691,829. As each of the references demonstrate, the approach those skilled in the art have generally taken is not to more accurately ascertain the appropriate time delay but rather to make that time delay as constant as possible. Thus, each of the references disclose feedback systems or monitoring systems which are designed to sense the location--not the time--at which droplet formation occurs. The system can then utilize that information as a feedback into the system to adjust that location to eliminate variations. While this type of approach may work well in some applications, it has a number of limitations. Significantly, these can include a failure to assure optimum drop delay timing in the first place. They also can be limited by the fact that the feedback systems themselves may act to move conditions within the flow cytometer away from optimum. As those skilled in the art readily understand, there are specific physical relationships which must exist for the flow cytometer to operate in an optimum fashion. The conditions should be set based upon the temperatures, pressures, frequencies, nozzle sizing, and the like for a given system. Any movement away from the correct relationships can negatively impact the resolution of the device. Thus, approaches which tend to apply corrections to counter the variations which may exist as a result of true conditions can represent an undesirable approach to solving the problem.
As mentioned, there has been a long felt and unsatisfied need for a device which not only automatically determines the optimum time delay for differential charging but also permits this time delay to be automatically ascertained during the actual processing of a substance. As the present invention shows, such a system is indeed possible and, in fact, can be implemented using arts and elements which have long been available. To some extent, apparently, solutions such as those of the present invention have not been achieved because those skilled in the art seem to have been directed away from the technical direction pursued in the present invention. This may have been the result of the fact that those skilled in the art did not truly appreciate the nature of the problem. It may also have been the result of the fact that those skilled in the art were misled by some of the presumptions and assumptions with respect to the types of systems which were appropriate for consideration. Although substantial attempts had been made at controlling a constant location for the formation of the drop, to some extent this may have only indicated that those skilled in the art failed to truly understand that the problem was not controlling the location but rather adapting to conditions as they may vary. The present invention shows that such an approach can be taken and that adapting to conditions actually experienced can be practically achieved. Until the present invention, a practical system which allowed for automatic determination of the exact time delay actually existing in the first place under true processing conditions was not available. Similarly, a practical system which has the ability to automatically ascertain the time delay and adapt to it during processing was not previously available.